磁微球为载体的H5N1 AIV免疫PCR检测方法分析

目的分析磁微球为载体的H5N1 AIV免疫PCR检测方法。方法模板选择pUC19,需要使用5’端标记的生物素作为引物,实现PCR扩增,进而制备有生物素的DNA分子。固相吸附载体选择磁微球,连接生物素标记的AIV H5N1血凝素蛋白和含有生物素的报告DNA分子,当两者结合之后,会将低含量病毒信号间接放大,建立起更加科学和可靠的检测H5N1 AIV免疫PCR方法。方法确定之后评价和分析PCR方法的灵敏性和特异性、最适报告DNA浓度等。结果采用免疫PCR检测方法可以成功检测到H5NI AIV病毒,确定报告DNA的最适浓度;此种方法也具有比较好的特异性和灵敏性。结论在建立免疫PCR检测方法的过程中以磁微球作为吸附载体,可以在检测H5N1 AIV时具有更高的灵敏性和特异性,是一种比较好的方法。     

免疫PCR检测HIV-1 p24抗原的初步评价

目的 对免疫PCR HIV-1 p24抗原检测体系的检出限、特异性和重复性等指标进行初步评价.方法 用建立的免疫PCR体系检测p24抗原,讨论检测方法的特异性、检出限和重复性指标,将空白对照的非特异性扩增条带用荧光密度值(FI)定量,初步提出特异性扩增下限的判定方法.结果 将非特异性扩增荧光密度值的(+3s)作为特异性扩增信号的判定下限,免疫PCR检测p24抗原的检出限为0.1 ng/L.结论 检测方法的检测限、特异性与重复性能满足HIV-1 p24抗原检测的需要.     

改良免疫-PCR检测肾综合征出血热特异性抗体

目的应用改良免疫-PCR方法检测肾合征出血热(HFRS)特异性抗体。方法在紫外线照射处理后的PCR管中进行免疫-PCR反应，以链霉亲和素作为连接分子连接生物素标记二抗和生物素标记DNA。PCR扩增DNA分子，琼脂糖电泳及扫描判断结果。结果改良免疫-PCR方法的阳性检出率为78．5％。常规免疫-PCR法和ELISA法的阳性检出率分别为62．3％和47．4％。结论改良免疫-PCR方法对出血热特异性抗体的阳性检出率高于常规的免疫-PCR法和间接ELISA法，可用于肾综合出血热持异性抗体的检测。     

夹心免疫PCR方法检测金葡菌肠毒素B

目的 建立双抗夹心免疫PCR的检测技术，用于检测极微量的抗原。方法 以亲和素作为连接分子，连接生物素化抗体和生物素化DNA，应用于免疫PCR中检测极微量金黄色葡萄球菌肠毒素B(SEB)。结果 双抗夹心免疫PCR的检测技术，检测SEB的灵敏度达0．1ng／L，与夹心ELISA方法比较灵敏度高10^3倍。结论 双抗夹心免疫PCR系统灵敏度高，可用于各种微量抗原的检测。     

PCR-金磁微粒层析法检测MTHFR基因C677T多态性评价

目的探讨PCR-金磁微粒层析法检测MTHFR基因C677T多态性进行方法学评价和性能确认,为个性化叶酸补充剂量提供参考依据。方法从检测下限、准确性、抗干扰能力、稳定性方面进行评价和确认。检测下限:分别选取MTHFR基因C677T野生型(677CC)、杂合突变型(677CT)和纯合突变型(677TT)外周血样本,提取基因组DNA,A260/A280在1. 65～1. 95之间,定量并稀释至2. 5 ng/μl、5 ng/μl、10 ng/μl三个浓度,每种基因型各浓度一式三份,然后进行PCR-金磁微粒层析法检测,统计三种基因型各浓度样本的检出率。准确性:将992例PCR-金磁微粒层析法检测的样本,提取基因组DNA,送第三方检测机构进行Sanger测序验证,比较二者的一致性。抗干扰能力:取三种基因型的外周血样本,分别加入终浓度为10 mg/ml的血红蛋白(Hb)、0. 2 mg/ml的胆红素(BIL)和5 mg/ml三酰甘油(TG),然后与不加任何干扰物质的样本同时进行检测,比较干扰组与正常组的检出情况。稳定性:取三种基因型的样本各1例,每种基因型各一式十份做平行检测,比较各基因型的检出情况。结果 (1)三种基因型在2. 5 ng/μl浓度水平均没有被检出,而在5 ng/μl和10 ng/μl浓度水平一式三份全部被检出,灵敏度为5 ng/μl。(2)992例样本的测序结果与PCR-金磁微粒层析法的检出结果完全一致,准确性100%。(3)三种基因型加干扰物的检出结果与不加干扰物的检出结果完全一致,抗干扰能力强。(4)三种基因型样本各1例平行检测10次的结果高度一致,稳定性高。结论 PCR-金磁微粒层析法可有效用于MTHFR基因C677T多态性检测,该法具有灵敏度高、准确性高、抗干扰能力强、稳定性高等特点,且无需特殊大型设备,具有快速、便捷、准确、经济等优势,适合在各级医疗机构开展。     

副溶血性弧菌的免疫捕获PCR检测

目的:将免疫捕获PCR法应用于副溶血性弧菌检测。方法:将脱毒后的副溶血性弧菌作为抗原免疫家兔,得到抗血清包被PCR管,对待检的副溶血弧菌进行免疫捕获,利用捕获到的菌体作为模板进行PCR检测。结果:PCR体系对副溶血性弧菌的tlh基因扩增具有特异性;抗体对副溶血弧菌起到一定程度的富集作用,包被抗血清后使PCR扩增出来的阳性结果更为明显,而抗血清本身没有扩增出DNA条带。结论:该检测方法能够对副溶血性弧菌进行特异性的检测,同时包被抗体的捕获对提高检测灵敏度起到一定的作用。     

免疫磁捕获-PCR检测大肠杆菌O157

目的 使用免疫BMPs,对牛粪标本中的大肠杆菌O157进行免疫磁捕获,再对捕获物进行PCR检测,研究免疫磁捕获-PCR检测牛粪标本中大肠杆菌O157的方法.方法 从磁性细菌中获取BMPs,将大肠杆菌O157抗体接种于BMPs表面,制成免疫BMPs.使用免疫BMPs,对大肠杆菌O157:H7浓度为1×103cfu/g的牛粪标本,进行免疫磁捕获,对捕获物以vt1(Vero毒素1基因)、vt2(Vero毒素2基因)为增扩对象,进行PCR检测.作为对照,相同牛粪标本,直接对牛粪标本以vt1、vt2为增扩对象,进行同样PCR检测.结果 相同牛粪标本,经免疫BMPs捕获处理,捕获物的PCR结果显示vt1及vt2基因阳性.而未经免疫磁捕获处理的牛粪标本,直接PCR检测的结果显示阴性.结论 对牛粪标本,利用免疫BMPs,可捕获目标抗原,同时还可去除牛粪中存在的PCR反应抑制物等影响因素,提高检测敏感度. 7浓度为1×103cfu/g的牛粪标本,进行免疫磁捕获,对捕获物以vt1(Vero毒素1基因)、vt2(Vero毒素2基因)为增扩对象,进行PCR检测.作为对照,相同牛粪标本,直接对牛粪标本以vt1、vt2为增扩对象,进行同 PCR检测.结果 相同牛粪标本,经免疫BMPs捕获处理,捕获物的PCR结果显示vt1及vt2基因阳性.而未经免疫磁捕获处理的牛粪标本,直接PCR检测的结果显示阴性.结论 对牛粪标本,利用免疫BMPs,可捕获目标抗原,同时还可去除牛粪中存在的PCR反应抑制物等影响因素,提高检测敏感度. 7浓     

HIV-1核心抗原p24基因的表达、纯化及抗原性分析

目的在原核系统中表达全长HIV-1 p24抗原,并对其抗原性进行鉴定。方法利用PCR技术从HIV-1全基因质粒(BH-10)中扩增p24抗原基因,通过酶切消化后连接到表达载体pET22b上,用此连接产物转化大肠埃希菌BL21(DE3),经IPTG诱导,表达p24抗原。运用双酶切技术、SDS-PAGE电泳检测插入基因片段的正确性,并用W estern B lot(WB)及ELISA法对表达产物的抗原性进行检测。结果PCR产物和构建的重组质粒pET22b-p24经双酶切插入的外源基因片段均为690 bp,与预期p24抗原全基因片段大小一致。纯化蛋白SDS-PAGE电泳,可见1条相对分子量约26×103的外源表达蛋白带,与预期大小一致,未见杂蛋白带。WB结果显示重组蛋白与HIV-1阳性血清呈特异性反应,与健康人血清没有反应。ELISA检测p24抗原灵敏度为93.94%(62/66),特异性为93.33%(28/30)。结论构建了HIV-1 p24表达载体pET22b-p24,并在原核细胞中高效表达,其表达产物具有良好的抗原性,为研制HIV抗体确认试剂奠定了良好的基础。     

HIV-1/2抗体和P24抗原联合检测方法的建立及应用

目的建立HIV-1/2抗体和P24抗原联合检测的酶免疫检测方法,用于HIV感染的实验室诊断,缩短HIV感染的检测窗口期。方法制备抗HIVP24单克隆抗体和多克隆抗体,联合使用基因工程HIV1/2型抗原和抗HIVP24单克隆抗体包被酶联反应板,以辣根过氧化物酶标记的HIV1/2型抗原和生物素化的兔抗HIVP24抗体作为标记物,建立联合检测HIV1/2抗体和P24抗原的ELISA方法,并对其特异性、敏感性和稳定性进行鉴定。结果检测P24抗原质控品的灵敏度可达0.2ng/ml;检测187份HIV阳性血清、210份其他疾病患者血清和520份正常人血清,其敏感性和特异度分别为100%和99.62%。同时重复12次检测4份HIV阳性血清,变异系数均小于10%。结论本方法可以在1.5h内一步同时检出特异性HIV-1/2抗体和HIVP24抗原,缩短了HIV感染的检测窗口期,具备快速、简便、特异、敏感等优点,适用于HIV感染的实验室诊断和流行病学调查。     

新型冠状病毒VSV假病毒的 构建及其感染能力的初步研究

目的 分析HIV-1感染者在长期抗反转录病毒治疗（antiretroviral therapy, ART）前后血浆中p24抗体和总HIV-1特异性抗体水平变化特点及其与HIV-1储存库关系。方法?选取2009年11月—2013年7月于我中心接受ART满5年的27例HIV-1感染者作为研究对象,采用荧光素酶免疫吸附试验和酶联免疫吸附试验,分别检测患者接受ART 0、1、3、5年时间点的p24抗体和总HIV-1特异性抗体的水平,同时对其与总HIV-1 DNA和细胞相关RNA（cell-associated RNA, CA-RNA）的水平进行相关性分析。结果?在ART 0、1、3、5年时,p24抗体水平分别为2.450（1.910～5.460）、1.460（1.080～2.160）、1.280（1.120～1.800）、1.570（1.090～2.180） LU;总HIV-1特异性抗体水平分别为3.378（3.157～3.710）、3.489（3.237～3.622）、3.446（2.742～3.573）、3.336（2.860～3.566）。p24抗体与总HIV-1 DNA和CA-RNA的水平均呈正相关（r分别为 0.407、0.305,P 均＜ 0.05）;总HIV特异性抗体水平与总HIV-1 DNA和CA-RNA水平没有相关性（r分别为0.155、0.165,P 均＞ 0.05）。结论?总HIV-1特异性抗体水平在ART期间没有明显变化,且与HIV-1储存库大小没有相关性,p24抗体水平随着ART时间增加呈逐渐降低的趋势,与HIV-1储存库的大小呈正相关,可为HIV-1特异性抗体和HIV-1储存库大小关系的研究提供依据。     

Calcium intake in the elderly: validation of a dietary questionnaire

Epidemiological studies of calcium and osteoporosis have been hampered by the lack of a suitable tool for assessing calcium intake. This report describes a new frequency and amount questionnaire for measuring present and past calcium intake in the elderly. The validity of the questionnaire was tested against two commonly used standards of dietary assessment, five-day duplicate diets and seven-day weighed dietary inventories. The resulting correlation coefficients were, respectively, r = 0.76 and r = 0.69, while that for repeatability was r = 0.84. Furthermore, the questionnaire categorized subjects into thirds of the distribution of intake with almost no gross misclassification. It is suggested that the present findings may be extended to the majority of normal, healthy elderly subjects, implying wide application for the questionnaire in the assessment of calcium intake in the elderly.     

Self-reported health problems among Swedish miners one year after unemployment

Unemployment is considered to be a public health concern sincedeterioration in the health of the unemployed is often anticipated.However, for some groups, such as miners, unemployment mightimprove health due to a cessation of potentially harmful occupationalexposures. This study evaluates the health of 79 miners in oneSwedish iron-ore mine, and 226 age-matched controls from thegeneral population, during one year after the closure of themine. The participants received a questionnaire regarding medicalhistory and subjective symptoms at the beginning of the studyperiod, and after one year. Statistically significant negativeeffects on self-reported health attributable to unemploymentwere not found, although neuropsychiatric symptoms were morecommon among the unemployed miners. The miners reported a statisticallysignificant improvement in grip force (p=0.031). They had asignificantly higher prevalence of symptoms associated withmining related exposures when compared with the population controls;pain in the upper extremities [relative risk (RR)=2.27, 95%confidence interval (Cl)=1.44–3.59), back pain (RR=1.84;Cl=1.237–2.75), vasospastic disease of the fingers (RR=2.05;Cl=1.18–3.57) and obstructive respiratory symptoms (attacksof dyspnea and wheezing: RR=3.67; Cl=1.167–11.6).     

Excès de cas humains et animaux de tularémie en France au cours de l’hiver 2007–2008 : émergence ou phénomène isolé ?

Context

Tularemia is a zoonosis affecting humans and hares in France. We describe the results of surveillance in both species, in 2007 and 2008.

Methods

Human tularemia cases are mandatorily notifiable in France since 2003. In hares, surveillance relies on volunteer hunter associations in all districts of the country. Data from mandatory reports and volunteer surveillance in 2007/2008 were analyzed and compared with previous results.

Results

In 2007/2008, 144 cases were reported in humans and 117 cases in hares. This was a 100% increase compared to previous years. Human cases differed from those of previous years only by the frequency of contact with breeding animals. Human cases without any documented risk exposure were also more frequent.

Conclusion

An increase of tularemia cases occurred in 2007/2008 in both species. Complementary studies are needed to identify the species reservoir in France to understand the causes of this peak of cases.     

Occupational health hazards in mining: an overview

This review article outlines the physical, chemical, biological, ergonomic and psychosocial occupational health hazards of mining and associated metallurgical processes. Mining remains an important industrial sector in many parts of the world and although substantial progress has been made in the control of occupational health hazards, there remains room for further risk reduction. This applies particularly to traumatic injury hazards, ergonomic hazards and noise. Vigilance is also required to ensure exposures to coal dust and crystalline silica remain effectively controlled.     

Manufacture and use of nanomaterials: current status in the UK and global trends

This paper provides an overview of the production and use of nanomaterials (NMs), particularly in the UK. Currently, relatively few companies in the UK are identifiable as NM manufacturers, the main emphasis being the bulk markets in metals and metal oxides, and some niche markets such as carbon nanotubes and quantum dots. NM manufacturing in the UK does not reflect the global emphasis on fullerenes, nanotubes and fibres. Some assumptions have been made about the types of NM that are likely to be imported into the UK, which currently include fullerenes, modified fullerenes and other carbon-based NMs including nanotubes. Many university departments, spin-offs and private companies have developed processes for the manufacture of NMs but may only be producing small quantities for research and development (R&D) purposes. However, some have the potential to scale up to produce large quantities. The nanotechnology industry in the UK has strong R&D backup from universities and related institutions. This review has covered R&D trends at such institutions, and appropriate information has been added to a searchable database. While several companies are including NMs in their products, only a few (e.g. manufacturers of paints, coatings, cosmetics, catalysts, polymer composites) are using nanoparticles (NPs) in any significant quantities. However, this situation is likely to change rapidly. There is a need to collect more information about exposure to NPs in both manufacturing and user scenarios. As the market grows, and as manufacturers switch from the micro- to the nanoscale, the potential for exposure will increase. More research is required to quantify any risks to workers and consumers.     

深圳公立医院管理体制改革制度设计

深圳公立医院管理体制改革实行政事分开、管办分开,在理事会架构下按法定机构模式组建市医管中心,落实公立医院运营管理自主权.作者从当前公立医院管理体制的弊端入手,介绍了深圳市进行公立医院管理体制改革的基本思路及改革方案设计的主要举措,深入剖析了的改革方案的特点,并对改革效果进行了预测.     

Effects of nitrogen dioxide on red blood cells of rats: Changes in components of red cell membranes during in vivo exposure to NO2

Red cell membranes, prepared from red blood cells of rats exposed to 4, 10, or 20 ppm nitrogen dioxide (NO₂) for 1 to 10 days, were examined for evidence of changes in membrane components. Appreciable changes were not found in contents of phospholipid and cholesterol during exposure to 10 ppm NO₂. By contrast, protein content altered with the time of exposure. Moreover, changes in protein composition were observed by employing sodium dodecyl sulfate — polyacrylamide gel electrophoresis. Twenty-four-hour exposure to NO₂ at the concentration above 10 ppm resulted in a marked increase in the percentage of lysophosphatidylethanolamine (LysoPE) to the total phospholipids. The prolonged exposure to 10 ppm NO₂ gave rise to a further increase in LysoPE, whereas the percentage of phosphatidylethanolamine (PE) showed a gradual decrease. A 1-day exposure to 4.0 ppm NO₂ also caused an increase in sialic acid content and decreases in those of PE and hexose. In addition to contents of these components the percentage of LysoPE increased 5 days after exposure and the elevated values were maintained up to the end of exposure period. These results demonstrate that red blood cells in circulation exhibit different membrane properties in terms of lipid and carbohydrate composition during 10 days of exposure to 4.0 ppm NO₂.     

Investigating clusters in the workplace and beyond

Clusters of disease are common and occur in the workplace and in the general community. They often arouse considerable concern among the population. Investigations have sometimes lead to exciting new knowledge, but in general the investigation of clusters is difficult and often unrewarding, especially for community clusters. In the workplace, investigations are more likely to find associations and even new causes, but still many clusters remain enigmatic. Despite this, there are many reasons for investigating clusters, including allaying community concern and identifying uncontrolled exposures. A structure for investigating clusters in the workplace is suggested.